The matricellular protein CCN5 prevents anti-VEGF drug-induced epithelial-mesenchymal transition of retinal pigment epithelium

Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.

EMT in ARPE-19 cells in vitro 15 , and that it also inhibits CNV and the accompanying EMT of RPE in vivo in a laser-induced murine model 16 .
In the present study, we show that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), can induce EMT in ARPE-19 cells in vitro and in the RPE of mouse eyes in vivo.We further show that co-treatment with the CCN5 protein ameliorates these adverse effects of anti-VEGF drugs in vitro and in vivo.We also demonstrate that blocking VEGF signaling pathway induces EMT in RPE and that this adverse consequence can be prevented by co-treatment with CCN5.

Results
Anti-VEGF drugs induce EMT in ARPE-19 cells ARPE-19 cells are human RPE cell lines that have been widely used for the study of RPE functions in vitro.The effects of anti-VEGF drugs in ARPE-19 cells have been controversial.Many previous studies have demonstrated that anti-VEGF drugs including Bev, Ran, and Afl have no effects on the viability and proliferation of ARPE-19 cells [17][18][19][20][21] , while other studies have reported that Bev induced EMT in ARPE-19 cells [22][23][24] .To address this issue, ARPE-19 cells were treated with various doses of Bev, Ran, and Afl for five days, followed by western blotting and immunocytochemistry (Fig. 1).When treated at doses equivalent to clinical doses (Bev, 0.312 mg/mL; Ran, 0.125 mg/mL; Afl, 0.5 mg/mL) all three anti-VEGF drugs had slight but inconsistent effects on ARPE-19 cells.However, when treated at doses equivalent to twice the clinical doses, all anti-VEGF drugs consistently induced the expression of EMT marker proteins, α-SMA and fibronectin, as well as a potent pro-fibrotic molecule, CCN2 within five days (Fig. 1B, C).Immunocytochemistry also showed a significant elevation in the expression of α-SMA with all anti-VEGF drug treatments (Fig. 1D, E).ZO-1, a tight junction protein, was used to demarcate individual cells (Fig. 1D).These drugs did not affect cell viability (data not shown).Altogether, these data suggest that all the tested anti-VEGF drugs have the potential to induce EMT in ARPE-19 cells.We previously showed that CCN5 inhibits TGF-β-induced EMT in ARPE-19 cells 15 .To examine whether CCN5 can also inhibit EMT induced by the anti-VEGF drugs, ARPE-19 cells were treated with Bev, Ran, and Afl in the absence or presence of the recombinant CCN5 protein (500 ng/mL) for five days (Fig. 2A).Consistent with our previous findings, CCN5 down-regulated the expression of CCN2 induced by anti-VEGF drugs.Moreover, the expression levels of α-SMA and fibronectin elevated by anti-VEGF drugs were normalized by CCN5 cotreatment, as shown by western blotting (Fig. 2B, C).Immunocytochemistry confirmed a significant reduction in α-SMA expression induced by anti-VEGF drugs upon CCN5 co-treatment (Fig. 2D, E).Taken together, these results indicate that CCN5 inhibits EMT induced by the anti-VEGF drugs.It is of note that CCN5 alone did not affect the expression of the EMT markers (Supplementary Fig. 1).

Recombinant CCN5 protein inhibits EMT induced by Afl in mouse eyes
We then examined the effects of Afl and CCN5 in vivo in mouse eyes.Bev and Ran were not tested in this experiment because neither of them possesses considerable affinities to mouse VEGF [25][26][27] .Afl was intravitreally injected into mouse eyes at a dose of 40 μg/eye.This dose is the one routinely used in other studies where the anti-neovascularization activity of Afl was evaluated in mice [28][29][30] .Afl was injected alone or in combination with recombinant CCN5 protein (40 ng/eye).The eyes were enucleated and the RPE/choroid complex was separated at 14 days post-injection (Fig. 3A).Immunohistochemistry of the flat-mounted RPE-choroid complex exhibited that the α-SMA expression was significantly elevated by Afl, which was significantly inhibited by the co-treatment with CCN5 (Fig. 3B).This finding was clearly illustrated by quantitation of the α-SMA-positive areas (Fig. 3C).
Immuno-staining with anti-ZO-1 antibody revealed that RPE cells became irregular and enlarged upon the treatment with Afl, and that this abnormality was significantly normalized by CCN5 (Fig. 3D).Mean cell numbers were significantly reduced by Afl, and this abnormality was prevented by the co-treatment with CCN5 (Fig. 3E).
Simiarly, mean cell size was significantly increased by Afl, which was prevented by the co-treatment with CCN5 www.nature.com/scientificreports/(Fig. 3F).These results indicate that the anti-VEGF drug, Afl, induces EMT accompanied by morphological changes of RPE, and that CCN5 can inhibit the fibrotic deformation induced by Afl.

Antagonists of VEGF receptors induce EMT and upregulate CCN2 expression in ARPE-19 cells
We further pursued to elucidate the mechanism underlying the anti-VEGF drug-induced EMT in ARPE-19 cells.
The VEGF signaling pathway is inherently active in ARPE-19 cells at baselines and is suject to further activation (Supplementary Figs. 2 and 3).ARPE-19 cells were treated with 5 μM of SU5416, an antagonist of VEGFR-1/2, or 10 nM of ZM323881, an antagonist of VEGFR-2 for 5 days (Fig. 4A).Western blotting demonstrated that CCN2 and EMT marker proteins, α-SMA and fibronectin, were up-regulated by the treatment with both VEGF receptor antagonists (Fig. 4B, C).Immunocytochemistry also showed an increase in CCN2-and α-SMA-positive cells upon treatment with SU5416 and ZM323881 (Fig. 4D, E).These results indicate that the inhibition of VEGF signaling pathways can induce EMT in ARPE-19 cells, which is correlated with the induction of CCN2 expression.

Knockdown of CCN2 inhibits EMT induced by Afl in ARPE-19 cells
The role of CCN2 in retinal fibrosis has previously been shown 31 .To test whether CCN2 is also involved in the anti-VEGF drug-induced EMT, ARPE-19 cells were treated with CCN2 siRNA in combination with Afl for five days (Fig. 5A).CCN2 siRNA completely blocked the induction of CCN2 by Afl.Furthermore, the Afl-mediated induction of α-SMA and fibronectin was significantly ameliorated by CCN2 siRNA, as shown by western blotting (Fig. 5B, C).Immunocytochemistry confirmed that CCN2 siRNA inhibited the induction of α-SMA by Afl in ARPE-19 cells (Fig. 5D, E).The effects of CCN2 siRNA were comparable to those of CCN5 in blocking the Afl-mediated induction of EMT (Fig. 5B-E).These results suggest that Afl, and most likely other anti-VEGF drugs, induces EMT by activating CCN2 signaling pathways, and that CCN5 inhibits anti-VEGF drug-induced EMT through the downregulation of CCN2 expression.

Discussion
VEGF is well known for its activity as a pro-angiogenic factor 32,33 .Thus, anti-VEGF therapies exhibit clear benefits for patients with nAMD.Besides the pro-angiogenic activity, VEGF displays protective effects in diverse tissues including neuronal cells.For example, VEGF protects hippocampal neurons from hypoxic damages 34 .In the eyes, VEGF was shown to act as an anti-apoptotic factor for photoreceptors 35 , Müller cells 36 , and RPE 37 .Considering the protective role of VEGF in the eyes, it is conceivable to observe that anti-VEGF therapies targeted for nAMD are often associated with accelerated deterioration of nAMD to geographic atrophy 38,39 .Another pressing issue is that a substantial portion of patients with nAMD do not respond to anti-VEGF therapies 40,41 .These findings necessitate the need for effective yet safer modalities for the treatment of nAMD.
In this study, we demonstrated that anti-VEGF drugs, including Bev, Ran, and Afl, induced EMT in ARPE-19 cells when treated at 2× clinical doses (Fig. 1).Clinical doses used in this in vitro study were deduced from the dosage of drugs administered in clinics and the average volume of human vitreous humor.No adverse effects of anti-VEGF drugs were reported in ARPE-19 cells in a number of previous studies.For example, Bev did not affect the viability of ARPE-19 cells when treated at 2.0 mg/mL for 24 h 20 .Ran and Afl were not cytotoxic to ARPE-19 cells when treated at 1.0 mg/mL for 24 h 19 .Bev, Ran, and Afl were not cytotoxic to ARPE-19 cells when treated at 0.3125 mg/mL, 0.125 mg/mL, and 2 mg/mL, respectively, for 72 h 18 .Bev, Ran, and Afl did not affect the viability and mitochondrial membrane potential of ARPE-19 cells when treated at the clinical doses for 24 h 17 .In contrast, adverse effects of these drugs were reported in several other studies.For example, Bev induced EMT in ARPE-19 cells when treated at 1.25 mg/mL for 24 h 22 .Similarly, Bev induced EMT in ARPE-19 cells by regulating Notch signaling when treated at 0.25 mg/mL for 24 h 42 .Bev affected the proliferation, phagocytosis, and membrane potential of ARPE-19 cells when treated at 0.25 mg/mL for 48 h 23 .Bev induced EMT in ARPE-19 cells by regulating the TGF-β1-SMAD2/3 signaling pathway when treated at 0.25 mg/mL for 48 h.Collectively, www.nature.com/scientificreports/ it appears that anti-VEGF drugs do not affect cell viability but do induce EMT and affect cell function when treated at clinical or higher doses in ARPE-19 cells.Consistent with this hypothesis, we did not observe any effects of the anti-VEGF drugs on cell viability at both clinical and 2× clinical doses but observed pro-EMT effects of the drugs at 2× clinical doses (Fig. 1).We further showed that blocking VEGF signaling by antagonists of VEGF receptors led to EMT in ARPE-19 cells (Fig. 4).Therefore, it appears that the adverse effects of anti-VEGF drugs shown in this and other previous studies might be due to a reduction of VEGF signaling below a hypothetical threshold by the drugs.
CCN2, also known as CTGF, acts as a pro-fibrotic factor in diverse pathological situations [43][44][45] .It was shown that Bev up-regulated CCN2 expression in ARPE-19 cells via Fc-FcR interaction 22 .In patients with proliferative diabetic retinopathy (PDR), CCN2 correlated positively, and VEGF negatively, with the degree of fibrosis 46 .Immunofluorescence microscopy revealed that signals for TGF-β2 and CCN2 were significantly intensified in PDR patients who received Bev therapy compared with PDR patients who did not receive Bev therapy 47 .In consistent with these studies, we observed that CCN2 is induced by the anti-VEGF drugs (Figs. 1, 2).We further found that the knock-down of CCN2 abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells (Fig. 5), which indicates that CCN2 is the key factor mediating the adverse effects, not just a bystander.
CCN5 is an anti-fibrotic factor in various tissues 13,48 .We previously showed that CCN5 is secreted from source cells via its amino-terminal signal sequence and is subsequently endocytosed by neighboring target cells, after which it enters the nucleus and functions as a transcriptional co-activator or co-repressor.We also showed that CCN5 directly regulates the expression of SMAD7 at the transcriptional level 49 , and Sabbah et al. showed that CCN5 functions as a transcriptional repressor of TGF-β receptor II 50 .CCN5 inhibits TGF-β-induced EMT in ARPE-19 cells 15 .More surprisingly, CCN5 reverses pre-formed EMT in ARPE-19 cells, as it was previously shown to reverses pre-formed fibrosis in hearts 14 .
Based on the results of this study, we propose that anti-VEGF drugs can be safer when it is administered in combination with CCN5.This study lays a foundation for the development of an efficient yet safer therapeutic modality for nAMD.